The carrier injection and charge transfer at interfaces in quantum dot light‐emitting diodes (QLEDs) are commonly evaluated based on the energy levels of different functional layers. However, the actual charge dynamics in the experiments are found to be very different from the common expectations. In this work, QLEDs using 2,2′′,2′′′‐(1,3,5‐Benzinetriyl)‐tris(1‐phenyl‐1‐H‐benzimidazole) (TPBi) or zinc oxide (ZnO) nanoparticles as electron transport layer (ETL) are studied by impedance spectroscopy. It was the first time to observe that the hole injection and electron injection start at different applied bias. In QLEDs with TPBi ETL, at an applied bias as low as 0.5 V, large amounts of holes have injected into the hole transport layer, while electron injection only occurs after the applied bias increased up to about 3.0 V. This is caused by the intrinsic accumulated negative charges in the quantum dot (QD) layer. The adverse impacts of the negative accumulated charges in QD layer are mitigated by replacing TPBi with ZnO. Hole injection and electron injection start at the same applied bias, or 1.7 V if ZnO replaces TPBi. Charge transfer and neutralization processes between QD and ZnO layers are adopted to explain the above results.